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Objectives and questions: Cellular senescence (CS) is an irreversible state characterized by permanent cell-cycle arrest, disordered autophagy, loss of function in mitochondria and resistance to programmed cell death (PCD). Senescent cells release a specific secretome marked by the secretion of inflammatory cytokines such as interferons (IFN), and extracellular matrix (ECM)-degrading enzymes that can trigger CS in neighboring cells. The contribution of CS to late-stage osteoarthritis has been established; however, its role in early OA (EOA) development remains unexplored. Additionally, the accumulation of senescent cells may negatively affect autologous cell-based therapies for EOA. Therefore, this study seeks to identify for the first time, differentially expressed genes (DEG) between EOA and focal cartilage trauma (FCT) using RNA-sequencing (RNA-seq) and to assess the involvement of CS pathways in EOA pathogenesis with aim of improving cell-based therapeutic approaches.

Material and methods: Primary chondrocytes were isolated from cartilage chips derived from defect cleaning during autologous chondrocyte implantation procedures, and clinically classified as either EOA or FCT. Bulk RNA-seq was performed by NovoGene GmBH, with nine donors per group (n=9) included in the final analysis following quality control with FastQC software. DEG assessment was conducted using NovoMagic platform, whilst gene ontology (GO) enrichment analysis, pathway analysis (PA) and gene network predictions were performed using gProfiler, Reactome and GeneMANIA respectively.

Results: In total there were 310 significant DEGs in EOA (log₂FC≥1, padj<0.05) compared to FCT samples (186 upregulated, 124 downregulated). GO analysis revealed that downregulated genes in EOA samples were linked to ECM organization and synthesis, while upregulated genes were associated with DNA helicase activity, mitochondrial gene expression and RNA processing. PA indicated enrichment of mitochondrial fission, cell-cycle checkpoint regulation and mitotic processes, including G1/S transition and chromatid separation. Further comparison with the CellAge CS gene database identified 20 upregulated and 12 downregulated CS-associated DEGs. These genes were predominantly connected to macroautophagy, DNA damage repair response and oxidative phosphorylation, implying disordered autophagy as a driver for CS in EOA. Principal component analysis and gene expression heatmaps showed tight clustering of trauma samples, whereas EOA samples exhibited greater heterogeneity, suggesting high biological variability in EOA. Network prediction suggested activation of IFN signaling as a potential downstream effect of the CS-related DEGs.

Discussion and conclusions: Collectively, these results indicate that CS-related mechanisms and PCD pathways may be active in EOA development, which might make prevention of CS a valuable target for mitigation of EOA symptoms. These findings will be further validated and following analyses will aim to identify potential pharmaceuticals that target chondrocyte CS mechanisms.